The proposed research focuses on the mechanisms of mitochondrial ATPase reactivation and inhibitor protein release which operates during the re-energization of intact mitochondria and submitochondrial particles (SMP) isolated from ischemic cardiac muscle. The first aim of the planned work is to establish conditions which will differentiate clearly between two putative, independently regulated sites of ATPase inhibitor-enzyme interaction, a fast-releasing regulatory site which is controlled primarily by the trans-membrane pH gradient, and a slow- releasing binding site and to show how the slow-releasing binding site is regulated. Our second aim is to examine possible conformational changes in the inhibitor protein (using a fluorescence probe covalently bound to the inhibitor) which may accompany both its rapid release from the regulatory site and its slow release from the binding site during the re-energization of SMP. Our third aim is to quantitate further the importance of the reversible inhibition of the mitochondrial ATPase in the control of tissue ATP depletion in situ in ischemic heart muscle. Data recently acquired in this laboratory suggest the existence of two distinct kinds of interaction between ATPase inhibitor protein and enzyme. One is a rapid, regulatory interaction which affects enzyme activity during re-energization and the other is a slower interaction which allows the physical dissociation of the inhibitor from the surface of the membrane into the soluble fraction. The amounts of inhibitor present on particles in our various protocols will be measured using our newly developed rat heart SMP ATPase titration procedure (1,2). The accuracy of this new procedure for the measurement of amounts of dog heart ATPase inhibitor on SMP has been verified by comparing it directly to an RIA developed especially for the measurement of the dog heart inhibitor (1). RIA procedures will be employed as well. The planned research will also address the question of the importance of ATPase inhibition in situ in mitigating tissue ATP depletion during myocardial ischemia. Our long term goals are to further characterize the interactions between the mitochondrial ATPase and its natural inhibitor and to quantitate further their pathophysiological significance in ischemic cardiac muscle.